The safety of candidate pharmaceutical compounds must be evaluated in pre-clinical testing prior to advancement to human clinical trials. This pre-clinical testing seeks to evaluate the safety of the compound on the various organ systems of the human body. Late stage pre-clinical testing often involves testing in animal models to assess the effects of the compound on the various organ systems. However, animal studies are expensive, and both physically and administratively labor-intensive. To ensure animal comfort and compliance with ethical standards, researchers must devise and maintain stringent animal housing and research protocols. It is therefore desirable to eliminate unsafe candidate compounds prior to reaching the animal testing phase, reducing the time and expense allotted to animal testing.
In vitro testing is often used in the earlier stages of pre-clinical testing to eliminate unsafe compounds prior to advancement to the later animal stages. For example, interconnected cellular networks of cardiomyocytes may be formed on a substrate for the testing of potential new heart therapies. Primary cardiomyocytes harvested from an animal, or animal or human stem cell-derived cardiomyocytes, form interconnected cellular networks when cultured on a cell culture substrate. The individual cardiomyocytes within a network are connected through gap junctions that allow ions to flow from one cell to another. This electrical connection allows an electrical action potential, which is first generated by a pacemaker cell, to propagate from one cell to the next.
Formation of an electrical action potential starts with a buildup of charge across a cell membrane. This buildup occurs spontaneously in cardiac cells, and more frequently in pacemaker cardiomyocytes than non-pacemaking cardiomyocytes. When the transmembrane charge reaches a threshold value, ions rush into the first cell (the depolarization phase). This triggers an action potential, which is a sharp influx of additional ions into the cytoplasm. The gap junctions distributed across the cell membrane allow ions to flow into neighboring cells, enabling the spread of the action potential.
Molecular processes within the cell tie the electrical action potential to the physical contraction of the cardiomyocytes. The propagation of the cardiac action potential across an in vitro cellular network, and the resulting contraction, resembles the propagation and contraction observed within the human heart and thus is often referred to as a “beat”. Many in vitro cardiomyocyte networks exhibit spontaneous beating, where each cardiac action potential propagation (and corresponding physical beat) is followed by a brief pause and then another cardiac action potential propagation and beat.
In vitro diagnostics allow researchers to analyze non-electrical properties of many types of cells, such as cell viability, density, and proliferation rates. However, electrically active cultures, such as cardiomyocytes, enable researchers to test additional properties related to electrical activity. For example, a cardiomyocyte culture may be assessed by the gap junction distribution, or degree of electrical connectivity between cells. This property may relate to the ability of a beat to be transmitted homogenously throughout a culture.
In another example, electrical measurements taken from a cardiomyocyte give researchers an indication of the cell health, quality, and level of maturity. For example, patch-clamp techniques provide measures of the action potential of an individual cell. A patch-clamp uses an electrode inserted into the cell membrane to measure transmembrane voltage. For a healthy cell, the cardiac action potential is initiated with a depolarization phase, where sodium rushes into the cell. The depolarization phase is followed by a plateau phase, dominated by the influx of calcium, where the cells remains depolarized, and ultimately a repolarization phase characterized by an outflux of potassium and a return to the starting transmembrane potential. Patch-clamp technologies can be used to detect abnormalities in the action potential within a single cell, which may point to functional problems. However, performing testing on individual cells is difficult and time-consuming. Furthermore, cardiomyocytes may behave differently when separated from their network, thus calling experimental results into question.
Other technologies, such as impedance measurement systems, can provide information about the physical beating of the cells, but do not reveal important functional information associated with the electrical action potential. Finally, optical imaging of the network electrophysiology can be performed using secondary voltage sensitive optical reporters. However, these protocols may be time consuming and cytotoxic, eliminating the ability to perform multiple experiments on the same culture.
Microelectrode arrays (MEAs) having a plurality of microelectrodes situated within each well enable researchers to measure signals from electrically active cells cultured on their surfaces. Herein, “microelectrode” and “electrode” will be used interchangeably. Cells are cultured across the array of electrodes within a well such that signals are detected from multiple electrically active cells, such as cardiomyocytes, simultaneously. These signals, called field potential signals, may change shape in response to the addition of a candidate compound to the cardiomyocyte culture. The changes may be used to evaluate the cardiac safety risk of a compound. Additionally, these measures may be used to develop and characterize new stem cell lines, to compare the electrophysiology of the cells to in vivo signals from native cardiomyocytes, and/or to evaluate in vitro models of disease.
However, data trends relevant to the experimental question may be masked by confounding variables. For example, a beat in a culture with below average cell density may be slower than normal. This could be incorrectly interpreted as an immature developmental state in a stem cell differentiation process. Electrically active cell cultures may have spatial variability across the culture. One region of the culture may beat more frequently, or a beat may be transmitted faster in certain regions. A culture may also have temporal instability. For example, the beat period (time between beats) may increase or decrease sporadically due to environmental conditions such as, for example, changing temperature. This could lead to unreliable data if, for example, the researcher were testing a compound meant to increase heart rate.
Arrhythmia analysis for an in vitro assay according to conventional techniques consists of identifying, in a binary sense, whether an arrhythmic event has or has not occurred during a defined analysis time window. In other words, the presence or absence of drug-induced arrhythmic events is assessed (without quantification) according to conventional techniques. Recent studies have aimed to provide an analog, or graded, quantification of the arrhythmia burden induced by a test compound. For example, in a recent study, the effects of a test drug on an in vitro culture of stem cell-derived cardiomyocytes was evaluated at multiple concentrations and the number of irregular beats was counted during a fixed time window. The concentration at which 20% of the beats were irregular was compared to the clinical Cmax drug concentration to quantify arrhythmic risk. Guo, L. et al., “Refining the Human iPSC-Cardiomyocyte Arrhythmic Risk Assessment Model,” Toxicological Sciences: An Official Journal of the Society of Toxicology, Vol. 136 (2): pp. 581-94 (2013). Additionally, in another recent study, multiple quantitative metrics of irregular beats were developed and a multi-parametric analysis was used to characterize the arrhythmic phenotype of test compounds. Gilchrist, K. H. et al., “High-Throughput Cardiac Safety Evaluation and Multi-Parameter Arrhythmia Profiling of Cardiomyocytes Using Microelectrode Arrays” Toxicology and Applied Pharmacology, Vol. 28(2): pp. 249-57 (2015).